Absence of Bands on Western Blot Film

[maprinzz]

Hello All,
Am currently having a problem in Western Blotting. Am investigating the expression of HIF-1alpha,in Insulinoma cells produced from Rabbit. The primary antibody am using is labelled "Protein G-purified Rabbit Anti-Human/mouse HIF-1alpha antibody and my secondary antibody labelled "Anti Rabbit IGn/HRP". My dilemma is if the absence of bands on my film be due to the fact that my cells are of Rabbit origin while the primary antibody is directed against "human/mouse" even though it was purified in Rabbit. Should it be "uman/Rabbit"then? All comments are welcomed.
Thanks

[Pete_2007]

Hi maprinzz,

There are many reasons why you don't get bands on a western film. B

But to start with, I think you identified a potential problem with the protein itself.

1) If you are using an antibody directed against human/mouse HIF1-alpha, first I ask: is your HIF1-alpha the rabbit version or the human/mouse version?? It sounds like you are expressing the human/mouse protein in these rabbit cells. If not, you wouldn't want to use a anti-human/mouse antibody on a rabbit antigen. The antigens might not be similar enough for good recognition.

If everything makes sense for number 1) above, I have some other questions...

2) Secondly, is 1o a mono or polyclonal? Thirdly are you doing SDS-PAGE or native? Are you over-expressing the antigen in these cells or just endogenous expression? Fourthly, and finally I ask are you doing blots on cell lysate or tissue? And is it whole cell protein extraction or nuclear protein extraction? If dealing with a transcription factor, you might want to consider doing nuclear extractions, as this will enrich you factor, giving a higher chance of success for detection by WB.

If all else above is okay then...

3) I would recommend toying with conditions a bit:
- try increasing amount of 1o antibody
- try increasing protein amount loaded on gel


hope this helps!

good luck.

Peter

[maprinzz]

Hi Peter,
Thanks for the response.
The confusing aspect of the labelling is the "Rabbit anti" in the name, "Protein G-purified Rabbit Anti-Human/mouse HIF-1alpha antibody, making it look like suited for my rabbit antigen (insulinoma cell). Well,

My secondary is a poly; polyclona swine Anti Rabbit IGn/HRP.
Am using SDS-PAGE. Am investigating if the HIF is induced by exposing the cells to DFX.
The blots are on lysates; protein extracted from these cells following exposure.
And finally, just as you said, the HIF is a keen transcription factor. My assignment now is to look for a protocol for nuclear protein extraction-ok.

I think I will try increasing the antibody conc. For the protein amount for loading, I am afraid there might not be an efficient separation if I apply more than the current 50 ug on my schedule; since am using the small sized gel (SDS-PAGE)from Invitrogen.

Thank you very much.
I will give you a feed back on how it goes.
Prince